Adaptive capacity to dietary Vitamin B12 levels is maintained by a gene‐diet interaction that ensures optimal life span

Diet regulates complex life‐history traits such as longevity. For optimal lifespan, organisms employ intricate adaptive mechanisms whose molecular underpinnings are less known. We show that Caenorhabditis elegans FLR‐4 kinase prevents lifespan differentials on the bacterial diet having higher Vitamin B12 levels. The flr‐4 mutants are more responsive to the higher B12 levels of Escherichia coli HT115 diet, and consequently, have enhanced flux through the one‐carbon cycle. Mechanistically, a higher level of B12 transcriptionally downregulates the phosphoethanolamine methyltransferase pmt‐2 gene, which modulates phosphatidylcholine (PC) levels. Pmt‐2 downregulation activates cytoprotective gene expression through the p38‐MAPK pathway, leading to increased lifespan only in the mutant. Evidently, preventing bacterial B12 uptake or inhibiting one‐carbon metabolism reverses all the above phenotypes. Conversely, supplementation of B12 to E. coli OP50 or genetically reducing PC levels in the OP50‐fed mutant extends lifespan. Together, we reveal how worms maintain adaptive capacity to diets having varying micronutrient content to ensure a normal lifespan.

| INTRODUC TI ON
The ability to forage on a wide range of diets is evolutionarily advantageous as organisms would flourish even when their preferred diet is depleted. Nutritional inputs play an indispensable role in maintaining cellular activities, and as a result in sustaining all life-history traits such as development and reproduction. Nutritional availability also has a significant impact on the health and aging of an organism (Maier et al., 2010).
Diet generally comprises of macronutrients but is also an important source for micronutrients such as vitamins that function as metabolic cofactors. As animals are exposed to food of various nutritional qualities, they have evolved intricate mechanisms to maintain homeostasis and normal life-history traits, including life span, in response to varied dietary cues, although the underlying molecular mechanisms are less explored.
Recent research has also started to elucidate how micronutrients regulate life-history traits of worms, including longevity (Bito et al., 2013;Bito & Watanabe, 2016;Maynard & Weinkove, 2020;Virk et al., 2012Virk et al., , 2016. This is primarily because the metabolically active intestinal microbiota of C. elegans, a major source of micronutrients, provides a relatively less complicated and genetically well-regulated model to analyze the direct as well as bacterial feed-mediated effects of diet on life span. Although C. elegans feeds on a wide range of bacterial diets, they can maintain normal life-history traits on most. This adaptive capacity to different diets is maintained by genes that have mostly been identified serendipitously (Maier et al., 2010;Mizunuma et al., 2014;Pang & Curran, 2014). When any of these genes are mutated, the worms fail to maintain homeostasis in various aspects of cellular physiology and start displaying altered life-history traits on one diet and not the others. These "gene-diet pairs" have been instrumental in our understanding of how the quality of food influences life span and health (Maier et al., 2010;Mizunuma et al., 2014;Pang & Curran, 2014).
Recently, we serendipitously discovered a diet-gene pair where a serine-threonine-specific kinase gene (flr-4) mutant displayed a dietresponsive increase in life and health span (Kobayashi et al., 2011;Take-uchi et al., 2005;Verma et al., 2018). The kinase-dead flr-4(n2259) [flr-4(−)] lives longer when grown on Escherichia coli HT115 but not on E. coli OP50. In flr-4(n2259) grown on HT115, the p38-MAPK pathway was found to be activated, leading to higher expression of cytoprotective (CyTP) xenobiotic detoxification genes through the Nuclear Hormone Receptor-8 (NHR-8) transcription factor. This suggested that flr-4(−) mutants become responsive to the presence of a molecule(s) in HT115 to mount a specific response, whereas wild-type worms can maintain homeostasis.
In this study, we identify Vitamin B12 or cobalamin as the micronutrient in E. coli HT115 that flr-4(−) responds to, extending life span and increasing stress tolerance. We show that that flr-4 (−) worms are more responsive to the higher Vitamin B12 content of HT115. Consequently, these worms have increased flux through the one-carbon metabolism and transcriptional downregulation of the phosphoethanolamine methyltransferase gene pmt-2 that is responsible for the biosynthesis of phosphatidylcholine (PC) from phosphatidylethanolamine (PE). We show that altering the levels of PC in flr-4(−) worms, either genetically or by diet supplements, activates cytoprotective genes, increases stress tolerance and life span through the p38-MAPK. Together, the serine-threonine kinase FLR-4 maintains adaptive capacity towards a diet of different Vitamin B12 levels to preserve normal life-history traits of C. elegans.

| Flr-4(n2259) is more responsive to Vitamin B12
We sought to identify the key regulators originating from the bacterial diet that positively influence the life span of flr-4(−) worms.
We have observed that several genes functionally linked to the one-carbon metabolism, for example, cysteine, taurine, methionine, glutamate as well as glutathione metabolism genes, are significantly upregulated in flr-4(−) grown on HT115 (Verma et al., 2018).
Importantly, two enzymes linked to the one-carbon metabolism (METR-1 and MMCM-1) require Vitamin B12 (B12) as a cofactor ( Figure 1a). Since worms depend on the bacteria for the supply of B12, we investigated if the differences in these micronutrient levels in HT115 and OP50 dictate the life span of the flr-4 mutant.
We directly quantified the levels of B12 in the OP50 and HT115 bacterial strains using a competitive intrinsic factor binding-based assay and found that HT115 has significantly higher levels of B12 compared to OP50 (Figure 1b). Like mammals, C. elegans are unable to synthesize B12 and depends solely on the bacteria for its supply (Maynard & Weinkove, 2020). Given the higher levels of B12 in HT115, we next tested whether internal levels of B12 in worms also vary accordingly. As expected, we observed significantly higher B12 levels in worms grown on HT115, compared to OP50 (Figure 1c). The levels were increased several folds more when the bacterial diet was externally supplemented with B12 (Figure 1b,c).
Using an alternate cell biology strategy, we assessed B12 levels in the flr-4(−) by monitoring the GFP expression of the Pacdh-1::gfp reporter strain. Acdh-1, along with ech-6, hach-1, hphd-1 and alh-8, is a part of a non-canonical B12-independent pathway that metabolizes one-carbon metabolism intermediate, propionic acid, a toxic intermediate, into acetyl CoA ( Figure 1a). As such, this pathway is activated in the absence or by low levels of B12; otherwise, propionic acid is converted to succinyl-CoA through the canonical B12-dependent pathway. The expression of Pacdh-1::gfp transcriptional reporter faithfully reports the levels of B12, with high expression corresponding to reduced, and low expression to high B12 levels in worms (Watson et al., 2014(Watson et al., , 2016. We grew Pacdh-1::gfp and flr-4(−);Pacdh-1::gfp strains on OP50 or HT115 and visualized GFP fluorescence. We found that acdh-1 expression was significantly high on OP50 compared to HT115, confirming our biochemical quantification that HT115 has higher levels of B12 ( Figure   S1a). The GFP expression was significantly suppressed when worms were grown on OP50 supplemented with B12. We also validated this by quantifying mRNA levels of acdh-1 and hphd-1 ( Figure S1b).
We found that HT115 has more B12 compared to OP50, but the B12 levels in the wild-type and flr-4(−) are comparable when grown on the B12-rich diets (either HT115 or OP50 supplemented with 128 nM B12). So, the phenotypes of flr-4(−) reported earlier, that is, increased CyTP expression and longer life span on HT115, may not simply be explained by the differences in the levels of B12 in the bacteria. The question would arise as to why wild-type worms also do not benefit from the higher levels of B12. One way of explaining the observation is by surmising that flr-4(−) may be more responsive to the levels of B12 in the bacteria. To address this, we compared the expression of acdh-1 and hphd-1 in wild-type and flr-4(−) grown on OP50 ( Figure S1b). We found that flr-4(−) have lower acdh-1 and hphd-1 levels on OP50, compared to wild-type, suggesting that these worms are more responsive to B12.
Responsiveness to B12 may also be measured by analyzing the ability of the wild-type and mutant worms to detoxify externally supplemented propionate when fed either OP50 or HT115. We grew the wild-type or flr-4(−) on increasing quantities of propionic acid, a toxic metabolite, on OP50 or HT115 diet and determined whether the worms are developmentally arrested, or their growth is slowed down.
We found that both wild-type and mutant worms grown on HT115 are more resistant to 110 mM propionic acid, compared to the worms grown on OP50, showing again that the latter has a lower concentration of B12 (Figure 1d,e). On further increasing the concentration of propionic acid to 150 mM, flr-4(−) worms on HT115 still produced viable offspring that developed through the larval stages while those on OP50 were mostly arrested at L1 (Figure 1d,e). These experiments show that the mutant worms are more responsive to the presence of B12 in HT115 and can benefit from it in terms of mounting a better propionate detoxification response through the canonical pathway.

| Vitamin B12 influences CyTP gene expression in flr-4(n2259)
Having established that HT115 has higher levels of B12 and that flr-4(−) is more responsive to it, we asked whether these attributes can explain various phenotypes where flr-4(−) differs from wild-type in a bacterial feed-dependent manner. We have previously shown that flr-4(−) worms have activated p38-MAPK pathway leading to increased expression of downstream CyTP genes that supports increased life span, only when fed HT115 (Verma et al., 2018). So, we asked whether supplementing OP50 with B12 will generate the same transcriptomic response. As a representative CyTP gene, we had earlier used the Pcyp35B1::gfp (CY573) transgenic line (Verma et al., 2018). We grew Pcyp35B1::gfp or flr-4(−);Pcyp35B1::gfp worms on HT115, OP50 as well as OP50 supplemented with different F I G U R E 1 Flr-4(n2259) is more responsive to higher Vitamin B12 content of Escherichia coli HT115. (a) A simplified view of the One-carbon metabolism in Caenorhabditis elegans, comprising of the folate, methionine and propionate (canonical and non-canonical) metabolic pathways. The metabolites are marked in black. The METR-1 and MMCM-1 enzymes use B12 as cofactors. (b) The B12 levels of OP50, HT115, OP50 supplemented with B12, BW25113 and ∆tonB. Data presented as mean value of six biologically independent experiments ± SEM. p-values determined by unpaired two-tailed t-test. (c) Diet-dependent increase in B12 levels in wild-type and flr-4(−) fed OP50, OP50 supplemented with B12, and HT115. Data presented as mean value of three biologically independent experiments ± SD. p-values determined by multiple t-test using the Holm-Sidak method. (d) Wild-type and flr-4(−) animals were subjected to propionate toxicity assay. L1 synchronized worms were grown on OP50 or HT115 supplemented with indicated concentrations of sodium propionate and checked for developmental arrest 72 h post-L1. With increasing concentrations of propionate, both wild-type and flr-4(−) worms fed OP50 showed developmental arrest. Upon feeding HT115 with increasing concentrations of propionate, flr-4(−) but not wild-type reached adulthood, suggesting that the flr-4(−) is more responsive to Vitamin B12. One of three biologically independent experiments shown. (e) Quantification of propionate toxicity assay in (d) using three independent experiments. Error bars are mean ± SD. p-value determined using two-way ANOVA with Sidak multiple comparisons test. Experiments were performed at 20°C. Source data is provided as a source data file.   Figure S2a,b).
To confirm that the increased CyTP response is indeed mediated by the B12 absorbed by the bacteria, we performed an additional experiment with the E. coli ∆tonB bacterial strain defective in micronutrient absorption (Revtovich et al., 2019). Since the ∆tonB mutant is in a different genetic background, we first tested the parent E. coli BW25113 strain for the Pacdh-1::gfp response.
We found that the expression of GFP was low, comparable to HT115 ( BW25113 and ∆tonB. We found that the expression of GFP was lower in ∆tonB compared to BW25113 and HT115, comparable to OP50 (Figures 2c and Figure S2c). This experiment shows that if E. coli is defective in the uptake of B12 from the media, it is not able to provide benefits to the flr-4 mutant. Overall, these results confirm that levels of B12 govern transcriptional upregulation of beneficial CyTP in flr-4(−).

The increased expression of CyTP genes in flr-4(−) grown on
HT115 is due to the activation of the p38-MAPK pathway (Verma et al., 2018). So, we asked whether the increased expression of GFP in flr-4(−);Pcyp35B1::gfp was due to p38-MAPK activation by B12. For this, we supplemented OP50, expressing either the control RNAi or the sek-1 (the p38-MAPKK gene) RNAi, with B12 and grew Pcyp35B1::gfp or flr-4(−);Pcyp35B1::gfp on it. We found that this increase in GFP fluorescence was abrogated on sek-1 RNAi; there was no difference observed in the case of Pcyp35B1::gfp (Figures 2d and Figure S2d). We performed pPMK-1 western analysis and found that B12 supplementation to OP50 led to increased phosphorylation  Table S1 for details). The wild-type worms had no effect on life span on B12 supplementation ( Figure S3d

| Enhanced flux through one-carbon metabolism is essential for CyTP gene expression, stress tolerance and longevity of flr-4 mutant
We have shown that the higher B12 levels in HT115 combined with increased responsiveness to the micronutrient in flr-4(−) leads to activation of the p38-MAPK and increased CyTP gene expression.
Since B12 is a major cofactor of the one-carbon metabolism that is an important source of diverse metabolites, flr-4(−) worms could benefit from the higher flux through this pathway when grown on HT115. We performed a metabolomics analysis and found that flr-4(−) responded to B12 by enhancing the flux through the pathway, while wild-type did not (Figure 4a). Interestingly, increased flux through One-carbon metabolism in HT115-fed flr-4(−) seems to induce CyTP expression as we found that knocking down metr-1 or was also suppressed on metr-1 and mtrr-1 (OP50) RNAi ( Figure S4a). Importantly, the genes of one-carbon metabolism were also re-

| Vitamin B12 supplementation may activate p38-MAPK in the flr-4 mutant by downregulating Phosphatidylcholine levels
Since B12 activates the flux through one-carbon metabolism as well as the p38-MAPK pathway in the flr-4 mutant, we investigated how these two pathways may be connected. It has been reported earlier that the depletion of Phosphatidylcholine (PC) levels activates p38-MAPK pathway, although the mechanistic details are less known (Ding et al., 2015). PC biosynthesis occurs via two routes; (1) from dietary choline through the Kennedy pathway and (2)  We used OP50 RNAi targeting cept-1, cept-2, cka-1, cka-2 and pcyt-1 that would prevent the conversion of choline to PC. The RNAi inhibition resulted in upregulation of GFP in flr-4(−);Pcyp35B1::gfp almost to the level of HT115 fed worms; however, no change was observed in Pcyp35B1::gfp (Figure 6a,b). Under any of these conditions, choline supplementation did not suppress flr-4(−);Pcyp35B1::gfp expression as it is not likely to be converted to PC ( Figure S6a). Importantly, knocking down of cept-1 or cka-1 in an flr-4(−) background, using the OP50 RNAi system, significantly increased osmotic tolerance and life span; no effect was seen in the wild-type (Figure 6c,d; Figure  S6b,c). Together, these experiments show that high dietary B12 may lead to lower PC levels which in turn affects the cytoprotective gene expression, osmotic tolerance and life span of the flr-4 mutant.

| Transcriptional downregulation of phosphoethanolamine methyltransferase gene pmt-2 on high Vitamin B12 diet
Finally, we asked how higher B12 levels in HT115 may potentially lower PC levels. Recently, the Walhout lab has elucidated a novel gene regulatory mechanism (the B12 mechanism II) driven by B12 (Giese et al., 2020;Figure 7a). In response to low B12 levels (as in OP50 fed worms), the flux through the one-carbon cycle is suppressed which lowers SAM levels. Low SAM levels activate the NHR-114 transcription factor, which then transcribes one-carbon cycle genes, as well as acdh-1. We hypothesized that a low flux through the one-carbon cycle may activate pmt-1/pmt-2 and increase PC levels, which would suppress p38-MAPK and CyTP expression. We grew flr-4(−);Pcyp35B1::gfp and Pcyp35B1::gfp on pmt-1 or pmt-2 (OP50) RNAi and found that GFP expression was increased ( Figure 7b, quantification in Figure S7a,b). We also quantified transcript levels of pmt-1 and pmt-2 by qRT-PCR. Importantly, we found that only the levels of pmt-2 were decreased on a higher B12 diet, while levels of pmt-1 remained unchanged (Figure 7c). Although B12 seems to affect mRNA levels of only pmt-2, perturbing any of the two methyltransferases (pmt-1 or pmt-2) may lower PC to activate CyTP expression. In line with these findings, knocking down pmt-1 and pmt-2 (using OP50 RNAi) increased osmotic stress tolerance as well as the life span of the flr-4(−) (Figure 7d-g, Figure S7c-f).
Further, knocking down pmt-2 or pmt-1 led to increased activation of p38-MAPK (Figure 7h-i). Together, our data suggest that the B12 mechanism II may transcriptionally modulate PC levels in the flr-4 mutants grown on a B12-rich diet, thereby increasing downstream supplemented with B12. The supplementation was not able to increase life span in the flr-4(−);sek-1(km4). Life span and osmotic tolerance assays were performed at 20°C. One of three biologically independent experiment shown. Life span summary is provided in Table S1. Summary of osmotic tolerance assay is provided in Table S2. Experiments were performed at 20°C. Source data is provided as a source data file

| DISCUSS ION
In this study, we have shown that the worms maintain the adaptive capacity to diets of differing micronutrient content, specifically B12, by preventing ectopic activation of the p38 pathway. Our data suggest that in the flr-4 mutant worms, improved responsiveness to B12 coupled to increased availability of the micronutrient on feeding HT115 leads to elevated flux through the one-carbon metabolism.
Also, higher B12 results in a potential lowering of PC levels due to transcriptional downregulation of PMT-2. Since FLR-4 is active in wild-type worms, ectopic activation of the p38-MAPK is prevented, and life span homeostasis is maintained. However, the kinase-dead  Table S1. Summary of osmotic tolerance assay is provided in Table  S2. Source data is provided as a source data file

flr-4(-)
(e) (d) In C. elegans, like other organisms, B12 inputs from diet and microbial colonization of B12-producing bacteria in the intestine regulate the flux through the one-carbon metabolism, comprising of the methionine and folate metabolism arms, as well as modulate the clearance of propionate (Bito & Watanabe, 2016;Watson et al., 2014;Zhang et al., 2020). Due to the central nature of the one-carbon metabolism, B12 is known to play a role in immune modulation, stress tolerance, fertility, development and memory (Bito et al., 2013(Bito et al., , 2017Candito et al., 2003;Pront et al., 2009;Revtovich et al., 2019;Schaffner et al., 2019;Verma et al., 2018;Watanabe et al., 2003;Yoshii et al., 2019;Zhang et al., 2020). While B12 deficiency affects pathogen response, lowers fertility, delays growth, and shortens lifespan (Bito & Watanabe, 2016;Ding et al., 2015), excess of the micronutrient accelerates growth, increases fecundity and shortens life span (Macneil & Walhout, 2013;. Thus,  Table S1. Summary of osmotic tolerance assay is provided in Table S2. Source data is provided as a source data file nmur-1 was previously shown to regulate life span dependent on food type; the mutant lives long on OP50, not on HT115 (Maier et al., 2010). NMUR-1 works in the sensory neurons and helps the worms differentiate between the two bacteria depending on the lipopolysaccharide structure. A mutant of the proline metabolism pathway, on the other hand, accelerates aging on OP50 by producing mitochondrial damage due to the build-up of reactive oxygen species and 1-pyrroline-5-carboxylate (Pang & Curran, 2014;Yen & Curran, 2016).
Intriguingly, the NMUR-1 receptor pathway was required by the alh-6 mutant to shorten the life span on OP50. We have identified FLR-4 as another gene that maintains adaptive capacity towards different diets (Verma et al., 2018). Interestingly, both ALH-6 and FLR-4 use mechanisms of longevity assurance that overlaps with that of dietary restriction. Recently, a mutation in the mitochondrial ribosomal gene mrpl-2 that mediates protein translation in mitochondria was found to activate the Unfolded Protein Response of the mitochondria (UPRmt) and promote longevity only when fed OP50. They also attributed this effect to the low availability of B12. It will be interesting to know whether this gene genetically interacts with FLR-4. Further, the rict-1 mutants show differential life span, metabolism and development on different diets. The rict-1(−) animals that were fed HT115 lived longer than wild-type, but on OP50, their lifespan was significantly reduced (Mizunuma et al., 2014). We have found that when grown on rict-1 RNAi, the flr-4(−) worms perish, suggesting a genetic interaction that needs to be characterized. Thus, a concerted effort needs to be made to identify more gene-diet interactions as well as study the mechanistic crosstalks between the published pairs. Several previous studies document inverse correlations between phosphatidylcholine levels and p38 activation. In C. elegans, activation of p38-MAPK on sams-1 knockdown is suppressed if phosphatidylcholine levels are upregulated by the addition of choline (Ding et al., 2015). In IMR-90 fibroblasts, Lysophosphatidic acid (LPA) analogues (S)-N-palmitoyl-norleucinol 1-phosphate [(S)PNPA] was found to induce apoptosis by specifically inhibiting PC biosynthesis, leading to p38 activation (Gueguen et al., 2002). Exogenous application of PC has been shown to have anti-inflammatory properties in human Caco-2 cells treated with tumour necrosis factor-alpha (TNFalpha), by inhibiting p38 signalling (Treede et al., 2007(Treede et al., , 2009). Our work also implicates the PC levels to lowering p38 activation. This raises a pertinent question as to how the PC: PE ratio may regulate the p38-MAPK pathway. The reasons for this negative correlation need to be deciphered in future.

F I G U R E 6
Reducing flux through Kennedy pathway activates cytoprotective genes and increases life span of flr-4(n2259) even on Escherichia coli OP50. (a) The expression of gfp in flr-4(−);Pcyp-35B1::gfp is increased when grown on control (HT115) RNAi, compared to control (OP50) RNAi. However, when these worms are grown on cka-1, cka-2, pcyt-1, cept-1 and cept-2 in the OP50 RNAi system, gfp expression was elevated. (b) Quantification of (a). Average of three biological replicates ± SEM. p-value determined using two-way ANOVA with Tukey's correction. ns, non-significant. (c) The osmotic stress tolerance of flr-4(−) grown on control (HT115) RNAi is higher compared to control (OP50) RNAi. However, knocking down cka-1 and cept-1 using (OP50) RNAi leads to better osmotic stress tolerance of the mutant even in the OP50 feed. (d) The life span of flr-4(−) grown on control (HT115) RNAi is higher compared to control (OP50) RNAi. Knocking down cka-1 and cept-1 using an OP50 RNAi system leads to longer life span even on the OP50 feed. One of three biologically independent replicates shown for all experiments. All experiments were performed at 20°C. Life span summary is provided in Table S1. Summary of osmotic tolerance assay is provided in Table S2. Source data is provided as a source data file Diet-gene interactions may provide an adaptive advantage to invertebrates like C. elegans, but such interactions may play important role in the pathophysiology of human diseases. For example, dietgene interplay may be the basis of the "copper phenotype" of sporadic Alzheimer's disease (AD) where a low copper diet reduces the risk of AD in individuals with altered copper metabolism (Squitti et al., 2014). Diet may also alter the impact of genetic variants on disease risk, as shown in the case of colorectal cancer where consumption of processed meat increased risk in individuals with rs4143094-TG and -TT genotypes (Figueiredo et al., 2014). Genotypes of individuals studied in some randomized clinical trials have also been found to modify the effect of dietary interventions on weight loss, weight maintenance, lipid profile, insulin resistance, and blood pressure (Qi, 2014). This underscores the importance of directed studies to unravel more diet-gene interactions and their underlying mechanisms, that are easier to study in model organisms.

| E XPERIMENTAL PROCEDURE
Detailed experimental procedure is provided in Appendix S1.
Caenorhabditis elegans strains were maintained at 20°C on  (Giese et al., 2020). Lower vitamin B12 levels in OP50 would lead to increased expression of methionine/SAM cycle genes. We hypothesized that this will also increase the expression of pmt-1 and/or pmt-2 (grey box). (b) Representative experiment to show that the expression of flr-4(−);Pcyp-35B1::gfp is increased on pmt-1 or pmt-2 (OP50) RNAi. Quantification provided in Figure S7a Table S1. Summary of osmotic tolerance assay is provided in Table S2. Source data is provided as a source data file   (Giese et al., 2020) ? span analysis, RNA isolation, QRT-PCR analysis, CyTP gene activation assay, western blotting analysis was performed as previously published (Chamoli et al., 2014;Verma et al., 2018). For Osmotic stress assay, L4 stage worms were exposed to 350 mM sodium chloride for 9 min and recovery was scored. For Propionate toxicity assay, L1 synchronized worms were placed on propionatesupplemented plates and development was observed after 48 and 72 h. Intracellular B12 was measured by a kit-based electrochemiluminescence immunoassay (ECLIA) using COBAS e411 Analyzer.

Intracellular metabolites for MS-based targeted metabolomics
were extracted using cold Actonitrile-methanol-water and data were acquired using a Sciex Exion LCTM analytical UHPLC system coupled with a triple quadrupole hybrid ion trap mass spectrometer (QTrap 6500; Sciex) in a positive mode. Relative quantification was performed using MultiQuantTM software v.2.1 (Sciex).
Cysteine and Homocysteine were measured by HPLC (Agilent 1290 Infinity II LC system).

ACK N OWLED G M ENTS
We thank all members of the Molecular Aging for their support,

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that supports the findings of this study are available in the supplementary material of this article. All data used in the manuscript is presented as a source data file.


s2fieldsofstudy: Biology Psychology

text:
Studying food entrainment: Models, methods, and musings

The ability to tell time relative to predictable feeding opportunities has a long history of research, going back more than 100 years with behavioral observations of honeybees and rats. Animals that have access to food at a particular time of day exhibit “food anticipatory activity” (FAA), which is a preprandial increase in activity and arousal thought to be driven by food entrained circadian oscillator(s). However, the mechanisms behind adaptation of behavior to timed feeding continue to elude our grasp. Methods used to study circadian entrainment by food vary depending on the model system and the laboratory conducting the experiments. Most studies have relied on rodent model systems due to neuroanatomical tools and genetic tractability, but even among studies of laboratory mice, methods vary considerably. A lack of consistency within the field in experimental design, reporting, and definition of food entrainment, or even FAA, makes it difficult to compare results across studies or even within the same mutant mouse strain, hindering interpretation of replication studies. Here we examine the conditions used to study food as a time cue and make recommendations for study design and reporting.

Introduction
Given the over-abundance of food in modern society, it is easy to lose sight of the importance of feeding to animals in nature and in sculpting our own evolutionary history. For most animals, food is extremely scarce and being wise to opportunities to eat is essential for survival. As such, the circadian system has evolved to receive a number of different stimuli-i.e., light, temperature, and food-to keep biological processes coordinated and allow for adaptation to ever changing conditions (1,2). Food is an often neglected zeitgeber, being outshined by light, which is more intensively studied and much better understood (3). The light entrainment pathway works via excitement of intrinsically photosensitive retinal ganglion cells that project to the suprachiasmatic nucleus (SCN), which is the major regulator of circadian rhythms (4). The SCN serves as a light entrained oscillator (LEO) and its near 24-h neuronal activity is so robust that it persists in explanted tissue for more than a year (5). Application of the same logic used to tease apart the neural circuitry and molecular clockwork of the LEO has not yielded great success when applied to studies of the food entrained oscillator(s) (FEO) (6). In this review, we describe attempts at delineating the circadian biology of food entrainment using a multitude of model systems, including extensive use of rodents, and given the Trzeciak and Steele . /fnut. .
wide range of methodology we make suggestions for improvements and considerations in methods and interpretation of data.
Model systems used to study food entrainment FAA research has a rich history with early published descriptions of the behavior dating back 100 years (7). In this time an array of organisms of varying complexities have been studied. Honey bees (A. mellifera) have been shown to entrain to restricted feeding schedules and exhibit phase differences in expression of cryptochrome2 (cry2) and period (per) with timerestricted foraging (8,9). By contrast, studies in D. melanogaster show that restricted food availability is not able to entrain activity cycles (10,11). Food anticipation has also been welldocumented in fish. This behavior was first explored in 1964, when a pre-meal peak in locomotion was observed in both largemouth bass (M. salmonides) and bluegill (L. macrochirus) (12). Other studies have followed suit, utilizing goldfish (C. auratus) (13,14) and zebrafish (D. rerio) (15, 16), among other species, to attempt to tease out an FEO separate from the LEO. Studies have demonstrated that fish align their activity periods with mealtimes; however, the presence of a FEO in fish that is separate and independent from the LEO is still an open question. Some unconventional mammalian systems have also been utilized; a recent study employed a pig model to show that ghrelin antagonist administration lowered FAA (17). While the bulk of research in the field has been conducted on rats and mice, alternative rodent models like Syrian hamsters (M. auratus), gerbils (M. unguiculatus), and the diurnal rodent A. ansorgei have been helpful in demonstrating the widespread nature of FAA and testing variables such as age and photoperiod (18,19). For example, Syrian hamsters may express lower FAA under restricted feeding conditions due to a stronger sensitivity to the masking effect of daytime light conditions (20)(21)(22)(23). Syrian hamsters have also been utilized for SCN ablation experiments to confirm that the SCN is not required for FAA in a rodent model aside from rats or mice (24).
Rabbits (O. cuniculus) naturally nurse their young once daily in the early morning hours, providing a natural system for studying FAA (25,26). The eyes and ears of rabbit pups remain sealed until postnatal day (PD) 10; in addition, while retinal projections to the SCN are present at birth, its response to photic input is not fully developed until PD12 (27). Furthermore, circadian cycling of clock genes Per1, Bmal1, and Cry1 in the SCN lacks a clear rhythm until PD45 (27). Even with the lack of rhythmic clock gene expression, young pups develop increased preprandial locomotor activity, altered expression of hormones such as ghrelin and corticosterone, and induction of c-Fos expression in several hypothalamic nuclei, including oxytocin neurons in the paraventricular nucleus (PVN) (28-31). These factors have made the rabbit pup an important model in which to explore the FEO with minimal interaction from the LEO.
Rats (R. norvegicus) have been a vital system for studying food entrainment and have been utilized for lesion experiments that provided many key negative results, ruling out structures of interest. Rats with lesions of the SCN lose their light:dark (LD) entrained rhythms when fed ad libitum, but maintain robust FAA when given restricted food access (32)(33)(34). These findings were repeated in mice (35), supporting the presence of a FEO separate from the LEO and that the SCN is not necessary for the development of food entrainment. Further ablation experiments have been conducted in rats in an attempt to find a region of control for FAA, showing that ablation of the olfactory bulb, dorsomedial hypothalamus, and thalamic paraventricular nucleus, as well as vagotomy, fail to prevent FAA (36)(37)(38)(39)(40)(41). Historically these studies have lacked consistency, both in methodology and results, and as such the existence of a single structure regulating food entrainment remains a theoretical possibility (42).
Due to their increased size and complexity, many experiments have been completed in rats that would likely not be possible with mice. In a recent study, rats were taught to press a lever for food and then subjected to restricted food access, using lever pressing as a measure of FAA; the lever pressing showed a preprandial increase, even in animals with SCN lesions (43). Rats have also been shown to be more attuned to entrainment by palatable meals, developing FAA to the presence of a palatable treat provided in addition to standard chow ad libitum (44, 45). Another advantage of rats is their increased size in comparison to mice, making them more suitable for meal withdrawal experiments, which helped to rule out "hourglass" timers for food entrainment (46). Withdrawal of food is a key element to proof of entrainment; however doing so in mice, which are much smaller and extremely metabolically active, causes rapid weight loss and protocols with this withdrawal period are often not approved by local research ethics boards.
Even within the laboratory mouse (M. musculus), there is substantial variation in genetic makeup that has been shown to affect any number of phenotypes (47). Most studies of FAA in mice are done on the C57BL/6 background, with many utilizing the J substrain from Jackson labs; however, the BTBR strain, which is a popular model of autism (48), was recently shown to have superior FAA compared to C57BL/6J (49). Moreover, phase shifting experiments also demonstrated that the BTBR strain was better able to adjust to altered light or meal delivery times when compared to C57BL/6J mice (49). BALB/C mice are believed to have weakly-coupled circadian oscillators controlling their light-based rhythms (50). When these mice were used to study FAA, they quickly developed high levels of FAA when compared to the C57BL/6J strain (51). The inbred 129S1 strain has been tested under 60% calorie restriction (CR) conditions and showed that they redistributed almost all high activity behaviors to the pre-meal window, displaying "exceptional" FAA (52). Historically, it has been shown that C57BL/6(J) mice have relatively weak FAA when compared to rats (35), which begs the question-why has this been the primary mouse strain utilized for studying FAA when other strains are available that are more attuned to scheduled feeding? We suggest that researchers continue to explore alternative strain backgrounds and consider using different strain backgrounds that demonstrate stronger FAA. Furthermore, prior research has found both major and minor variations in the genomes of commonly used strains of mice, such as C57BL/6J (B6/J) and C57BL/6N (B6/N). For example, a common mutation in nicotinamide nucleotide transhydrogenase (Nnt) gene, which is found solely in the B6/J strain, appears to negatively impact mice in metabolic studies (53). Mice with the Nnt mutation have an increased chance of diet-induced obesity due to glucose intolerance, as Nnt is necessary for the maintenance of ATP synthesis (53, 54). On the other hand, B6/N mice carry a frameshift mutation in retinal degeneration 8 (rd8) and in the cell polarity complex component (Crb1), impairing vision (55). The effects of these particular mutations have not been studied for their effect on FAA.
Pendergast and Yamazaki (6) provided an authoritative review of food entrainment studies of mouse mutants, summarizing every knockout (KO) mouse tested (6). As a thought experiment, we describe the idealized mouse mutant that we have hoped to discover through genetic approaches to studying food entrainment. Firstly, this mouse would have normal light entrained rhythms and its total activity levels would be unaltered. Secondly, both its feeding, body weight, and body composition would be indistinguishable from controls. Third, its digestive and nutrient absorption system would be fully intact. Finally, it would have a complete deficit in predicting scheduled mealtime at a behavioral level-both when fed in the light cycle, dark cycle, and under constant conditions (DD or LL). Peripheral oscillations, such as liver gene expression, corticosterone secretion, and body temperature would all show normal entrainment. Whatever gene deletion is present in this mouse would be recapitulated in a conditional KO where this gene was removed from only a subset of neurons, indicating a brain region(s) that serves as the FEO. Alas, we are lacking such a mouse, but important strides have been made in this direction, including a mouse mutant lacking Nr1d1 in neurons that cannot predict scheduled mealtime when fed during the day (56).

Food entrainment methodology in rodents
Since we study FAA in rodents, we are focusing on this system here but the methodological issues described herein can be applied similarly to other organisms. For studies of food entrainment, experimental conditions vary considerably. For example, temporal restriction (RF) of feeding is the most common method, but the duration of food availability has a large range. Similarly, some studies offer a larger temporal window of food availability that tapers down over time while others do not. Calorie restriction (CR), feeding anywhere between 60 and 80% of normal food intake, is another commonly employed method for inducing FAA. CR has the advantage of fewer handling steps (one feeding vs. delivering and removing food) but the disadvantage is that mice can potentially "ration" their food and may not consume it all during a tight window (we have not observed this to be the case after the first few days of CR). Despite these concerns, a side-by-side comparison of body temperature and FAA in mice on 60% CR vs. 3-h RF showed similar entrainment of activity and body temperature to feeding (57). Multiple feedings can also be employed: for example, Luby and colleagues demonstrated that mice can anticipate several meals per day but not with high precision while Petersen et al. demonstrated that rats are much better at anticipation of multiple mealtimes (43,58).
Regarding RF, the temporal window of food access and when that occurs during the LD cycle varies a lot from study to study. At one extreme would be studies like that of del Rio-Martin et al.
(59) that fed mice during the entire 12 h dark cycle, leading to minimal FAA in controls and the erroneous conclusion that a mutant mouse (Pitx3 ak ) failed to show metabolic or behavior entrainment to scheduled feeding (59). When we studied the same mutant under a CR feeding regimen for many days (>40 of 60% CR), we observed robust FAA in the mutant and controls, However, by 2 weeks of timed 60% CR, there were no differences between groups; moreover, the orexin KO showed resistance to weight loss on CR, making it difficult to interpret this delay in establishing FAA. Another important point discussed by Pendergast and Yamazaki (6) is the use of fasting days both pre-and post RF. For example, fasting a mouse for 24 h prior to beginning RF reduces both fat and lean mass and leads to much more rapid appearance of FAA, even within a few days (63). Some laboratories even combine temporal and calorie restriction (64). Finally, it will be important to examine the effect of repeated testing on the same animals for parameters of food entrainment. For example, LeSauter and colleagues concluded from their studies of D2 dopamine receptor overexpression transgenic mice that this manipulation lowered "motivation" for food, but in their studies they repeatedly tested the same mice in a short-(4 h), medium (6 h), and long-duration (8 h) RF schedule, finding that only under the longest duration RF that the D2 transgenic mice had less FAA (65). Between rounds of RF, .
/fnut. . ad libitum access to food was given for variable amounts of time, ranging from 7 to 10 days but also including a fasting day in one of these respites (65). Expression of FAA should not necessarily be interpreted as lower motivation and it is important to apply instrumental assays to test this more explicitly. Does restriction amount or the amount of weight loss matter? In a study using C57BL/6J male mice, we compared 60 vs. 80% CR fed in the middle of the light cycle (66). Both the 60% and 80% CR groups demonstrated FAA within 7 days of scheduled feeding, losing about 10% body weight during that first week and weight loss continued to decline for both groups until day 14. However, the 80% group's body weight rebounded slightly as the study progressed and their level of FAA declined after day 14, whereas weight loss in the 60% group continued and the magnitude of their FAA was almost twice that of mice on 80% CR at later time points when both groups had clearly developed FAA. Thus, the amount of restriction and accompanying weight loss appeared to dictate the amount and maintenance of FAA. However, and quite unexpectedly, examining individual mice within either the 60% or the 80% group showed that there was no correlation between the amount of weight loss experienced by each individual mouse and the magnitude of FAA that it expressed. Thus, weight loss or at least having mice in a negative energy balance appears to be an important aspect of obtaining maximal FAA (i.e., 60% has greater FAA than 80% after day 14), but at an individual level the weight loss is not predictive of the amplitude of FAA. In the future, a study that explicitly tests the effect of starting body weight on food entrainment would be of interest. For example, a cohort of same-aged mice could be either over-fed, under-fed (without a timing component), or normally fed prior to entering a timed feeding study. This would allow for comparison of overweight, normal weight, and under-weight status on induction and maintenance of FAA. It is important to note that some laboratories distinguish between hypo-and normocaloric food entrainment and obtain some measure of food entrainment by scheduled feeding that does not result in a net negative energy balance (67).
Diet plays a large role in metabolism and has effects on feeding behavior. The ability to achieve FAA on specific diets corresponds with a macronutrient restrictive food anticipatory study in which subsequent restriction of protein, carbohydrate, and fat nutrients in rats did not substantially affect locomotor behavior prior to reward stimulus (68). Binge-eating behaviors can lead to obesity and often stem from not only the addictive behavior of eating at a specific time during the day but even just from the sight or smell of palatable food (69). In the past, Mistlberger et al. attempted to observe the potential effects of food in relation to activity in mice by testing rats on two different types of diets. The main difference between these two diets was the lack of a simple sugar in unformulated or standard mouse chow, whereas formulated chow had sucrose. Rats in this study were deprived of either protein, carbohydrates, or high fat diet for about 2 weeks, then introduced to the missing macronutrient and tested for locomotor behavior using tilt cages (68). In other words, a palatable meal offered during caloric restriction is sufficient as an entrainment cue for food anticipation. These findings suggest an independence exists between a single macronutrient and FEO(s), thus diet in itself is not a sufficient cue to entrain any FEO(s).
High-fat palatable meals and "treats" have been shown to be a strong promoter of FAA. Palatable meals are loosely defined as being nutrient-rich with early studies utilizing a "nutrient-rich palatable mash" (45), but have included a variety of formulas such as high-fat rodent diets and treats like chocolate and cheese containing varying levels of sugar and fat content (52, 70). Overall, rats and mice have been shown to develop FAA and binge-type eating with the addition of high-fat meals, with correlating c-Fos activation in reward centers of the brain (44, 52, 71). A recent study also showed that, in rats, a high-fat diet stimulated higher c-Fos expression, stronger FAA, and more binge-type eating than a high-sugar diet (72). That being said, restricted access to a high-sucrose meal is known to activate c-Fos expression in the prefrontal cortex, lateral septum, nucleus accumbens and anterior lateral hypothalamus during the premeal window (73). It would be of interest to test whether the palatability, sweetness, and formulation of the diet would influence food entrainment, in particular the amount of FAA.

Factors other than food
Aside from diet and feeding times, there are a number of other variables that have been shown to impact FAA but are not always reported or considered. Overall, sex differences in FAA in rodents are modest, but should be studied and reported. Mice on RF schedules indicate clear differences between the sexes in regard to food intake, body weight, and food anticipation (61). This experiment, which separated male and female mice, depicts males to be generally more active and to have exhibited greater wheel-running activity during RF (61). Another study focused on sex differences in D1 receptor (D1R) KO mice also showed that female D1R KO mice had more severely attenuated FAA than the D1R males (74). In addition, ghrelin levels, the hormone responsible for triggering an appetite, were analyzed to be higher in female than male mice (61), which suggests gonadal hormonal differences can account for sex-related differences in regards to FAA. Supporting this, gonadectomized mice were observed to have similar food anticipation levels over a span of 10 days of RF (61). However, a series of follow-up experiments opposes this finding that differences in FAA are related to gonadal sex hormone differences. Instead, Aguayo et al. showed that singular manipulations to gonadal hormones, sex chromosomes, and developmental patterning in WT mice are not enough to explain the sex differences (75). This study used gonadectomized mice, sex chromosome copy mutants, and masculinized female mice that were treated with 17-β .
/fnut. . estradiol during their neonatal period and still reported no differences between males and females while on a calorierestricted feeding paradigm (75). Therefore, while there are reports that indicate male mice to be generally more active than females, gonadal hormone differences alone cannot account for these findings. We showed that sex difference in FAA was actually age dependent. Female mice that were 9-11 months old no longer had a decreased level of FAA compared to male mice (75). In addition to the loss of sex differences in mice aged 9-11 months (75), age has also been shown to reduce amplitude and onset of FAA. A study comparing FAA in young and aged rats showed that rats aged 24-25 months, being of extreme old age, had a longer onset time and a lower amplitude of FAA when compared to rats aged 3-21 months (76). Another study examined FAA in dysrhythmic rats aged 13-18 months compared to young control rats and young SCN-lesioned rats; here, they found that the aged rats were able to develop FAA and successfully entrain to a restricted feeding schedule, although aged rats with intact LD cycling were not run in concurrence to compare onset and amplitude of activity (77). As it has been shown that aged mice display a decreased capacity for reentrainment to LD phase shifts (78) it would stand to reason that the same would be true for entrainment to feeding schedules.
Manipulations in photoperiod have shown that the duration of the light cycle has a minimal effect on FAA and that the behavior is able to persist in both constant conditions (both LL and DD) (79, 80). Mice have also been tested in long-and shortday photoperiods, but neither consistently affected FAA (81). Interestingly, skeleton photoperiods enhanced FAA though an unknown mechanism (82).
Numerous studies of the gut microbiome have shown that time-restricted feeding or addition of specific microbiome derivatives (83) are capable of altering expression patterns of core clock genes both in vitro and in vivo [reviewed in Dass and de Ross (84)]. It has been shown that mice given standard chow ad libitum exhibit cyclical expression of gut microflora and that ad libitum access to high fat diet reduces these cyclical fluctuations; however, animals given time-restricted access to the same high fat diet retain the cyclical expression of the microflora (85). Another study demonstrated that disruption of LD rhythms by regular phase shifts resulted in changes in intestinal microflora only when mice were fed a high-fat diet, while mice fed normal chow ad libitum did not experience a similar change (86). In this study, it was discussed that a secondary stressor on top of phase shifting, such as alcohol consumption (87), high-fat & high-sugar diet, or general stress is often required to induce these microbiome changes and they may not occur in an "unchallenging" environment (88). While neither paper examined the gut microbiome of mice on a restricted feeding schedule of normal chow, given the stress likely induced by single housing and food restriction regimens, it would follow that such conditions may also induce changes in the gut microbiome of animals undergoing a "typical" FAA experiment, some of which may be cyclical as seen by Zarrinpar et al. Given the known interaction of the microbiome and expression of clock genes in peripheral tissues [reviewed in Dass and de Ross (84)], more investigation of how the microbiome affects behavior and FAA may be warranted. Initial studies could measure FAA in germ-free mice to establish the necessity of gut microbiota for food entrainment.
Other factors to consider are the activity measurement method and cage environment. For example, we observed that higher ambient temperature is suppressive of FAA (66) while others have obtained robust inductions of FAA and other readouts of entrainment at thermoneutral temperatures in mice (29 • C) (89). There are a variety of methods utilized for quantification of food entrainment, including but not limited to wheel running, lever pressing, telemetry sensors, photobeams, motion sensors, and video-based homecage behavior analysis (40, 43, 52, 60, 90, 91). Direct comparison between telemetry sensors and motion sensors have shown that quantification through these methods are equivalent (92). Alternatively, the inclusion of a running wheel in a cage has been shown to increase FAA; even mice with previous wheel-running experience showed increased FAA when given a locked running wheel, although mice without prior wheel-running failed to show a similar increase (90). It was also shown that circadian organization, observed by Per2:Luc expression, is affected by wheel running. Phase-shifting of the lung, spleen, and liver was observed and shown to be wheel-running specific, as an enriched environment alone did not produce the same effect (93). A major setback of a number of behavioral analysis systems is the requirement of single housing the mice in a barren cage. One example of this is in Gallardo et al. (66), where video-based homecage behavior is analyzed using the HomeCageScan software (behavioral definitions described in 88). This method allows for a more detailed breakdown of behaviors than simple activity sensors or photobeam breaks, which has shown to be useful in certain mouse mutant lines. For example, in leptin KO mice we observed an increase in preprandial hanging and jumping behavior but walking was not increased (57). The sum of high activity behaviors was not significantly increased in leptin KO mice on CR, because this measurement is dominated by walking behavior. We concluded that the leptin KO mice had an intact FEO because of their timing of jumping and hanging, but other groups have reported that leptin negatively regulates FAA, making their measurements using locomotor activity via photobeam breaks (94). In this study leptin KO mice had enhanced FAA, consistent with studies in rats that harbor a homozygous null mutation in the leptin receptor gene (95). Administration of leptin blunted FAA in leptin KO mice but leptin administration in wildtype mice had no effect on FAA (94). That being said, there are downsides of video recording behavior that should be mentioned: most video-based methods require an empty cage .
with minimal bedding, depriving the animals of enrichment (96). In Pendergast et al. (93), three separate measures of activity were taken by way of infrared motion sensors, wheel running, and infrared video analysis of eating behavior. This use of multiple techniques provides several measures of behavior while also allowing for an enriched environment for the animals (93). Given that FAA is likely an adaptation of animals to forage during periods of food availability, providing more forms of enrichment to allow for those behaviors may be ideal. Furthermore, studying food entrainment in socially housed animals has not been conducted to our knowledge, as most measures of activity require individual housing.

Recommendations and considerations for food entrainment studies
Based on the considerations and methodological issues described above, we advocate for the collection, presentation, and analysis of food entrainment data as follows: • Food intake values should be reported as raw and/or normalized to body weight. These feeding measurements should be reported prior to restriction and during, if appropriate (i.e. during RF it is possible to measure food intake whereas during CR the value is fixed).
• Although on its own, body weight loss is only loosely predictive of FAA, it is still important to report both as raw data (i.e. grams) and as a percentage of starting weight. Ideally, measurements of lean and fat mass would be presented as well, especially in cases of mouse mutants that have adipose or metabolic phenotypes (57). For example, it was reported that orexin KO or orexin neuron ablated mice had less FAA (62, 97), but when we tested timed CR in orexin KO mice we observed that they were resistant to weight loss during CR and eventually developed comparable FAA to controls (57). Since starting body weight and/or fat stores can be affected by genetic manipulations and will likely affect the development of FAA, these data need to be considered and reported.
• With respect to metabolism, measurements of indirect calorimetry (i.e. respiratory exchange ratios) are desirable especially when mutations cause metabolic phenotypes.
We are only aware of a couple of examples of studies of food entrainment that measure energy expenditure (60, 98) and recognize the difficulty in conducting them given the cost of obtaining the equipment. More studies of respiratory exchange ratios and energy usage under different feeding protocols and in different mouse mutants may reveal previously unappreciated principle components of food entrainment.
• Raw and normalized behavioral activity data should be reported. This will allow for i) visualizing total activity levels, which are often affected by gene deletions, and ii) for comparison to nocturnal activity peaks as a frame of reference. Thirdly, normalization to total activity corrects for differences within lines of mice and also allows for comparisons between studies (i.e. a 30% redistribution of activity to precede scheduled mealtime). In our study of sex differences in FAA using C57BL/6J mice, we observed no difference in premeal high activity behavior when comparing males vs. females, but when we normalized these data, the females clearly showed less FAA and higher nighttime activity (75).
• As recommended by Pendergast and Yamazaki (6), if permitted by the Institutional Animal Care and Use Committee, include at least one fasting day or meal omission after FAA has been established in order to discern if the mutation/manipulation in question is affecting the expression of FAA or the FEO itself.
• Conduct initial studies of FAA in 12:12 LD conditions with feeding in the middle of the light cycle. This can be followed by testing FAA for nighttime feeding as some mutant mice have specific deficits in FAA only for light cycle feeding [for example, see Podyma et al. (63)]. Finally, examining FAA in constant darkness or constant light and/or meal shifting experiments are an important third and fourth lines of inquiry that allow for studying FAA with less contribution from the LEO.
• Conduct longer duration experiments or do a fasting day(s) prior to timed feeding for shorter duration experiments. Studies of FAA range from less than a week (63) on the low end, to at least 2 months on the high end (60, 64). As starvation increases activity acutely (52), it is important to distinguish whether mice have a phenotype relating to hunger induced activity vs. food entrained activity, thus adding a week of RF/CR to a study can assist with interpretation of results in combination with body weight data (i.e. when weights have plateaued).
• Report strain information and track genetic purity of inbred rodent models to monitor genetic background and possible interacting alleles, especially in the context of KO/Cre transgenic mice made from ES cells on different genetic backgrounds where linkage disequilibrium makes it nearly impossible to obtain alleles on a pure background via backcrosses (47).
• Report lab diet formulations. As discussed above, foods with higher fat content tend to be suppressive of FAA while perplexingly, the palatable treats used to induce FAA in mice need to have high fat content (52, 70). It is also an outstanding question as to whether formulated diets will lead to more consistent or stronger food entrainment.
• Report sexes and ages of animals. Females are understudied in circadian biology and metabolism (99). The age of animals  102). Follow-up studies using modern neurogenetic techniques and/or more refined deletion techniques, using viral vectors in combination with single or dual recombinase systems, could yield greater insights into the FEO using these initial observations as inroads should they prove reproducible. Furthermore, unclear or conflicting results from broad lesion experiments could be further refined with more detailed analyses of these brain regions. For example, c-Fos staining in the supraoptic nucleus and main body of the PVN in the rabbit and rat brain showed activation after feeding, while the posterior PVN showed activation prior to the scheduled feeding time (103, 104). These smaller subregions are more difficult to target and may be treated as a single, larger "region" under less precise techniques.
• Come to a consensus on a more rigorous definition of "food anticipatory activity", and be more clear about whether FAA is reduced vs. eliminated. Could the field agree on a mathematical definition or a heuristic rule for defining FAA? For example, conduct a one-way ANOVA comparing pre-and post-restriction with a significant effect of time?
Or a simple rule such as the amplitude of the FAA peak must be at least half that nighttime activity peak to qualify as FAA? (52, 105). Other ideas to consider would be defining the start of FAA to be when activity surpasses the mean, or when an "acceleration" of a certain magnitude occurs (change in activity from previous bin). Given that most researchers collect food entrainment data in different ways, perhaps fitting a logistic curve to data could be useful since there is an upper limit to the amount of FAA that can be expressed. This objective will require a board consensus perhaps assisted by a metaanalysis of FAA or other food entrainment data. Finally, we need to appreciate the difference between a loss vs. a reduction in FAA or other metrics of entrainment. When FAA is reduced, it does not necessarily follow that the timing mechanism, or the FEO, is not working. As we collect more examples of mutations and manipulations that lower FAA, it will be important to measure other aspects of entrainment and potential masking effects (i.e., studies under DD, for example). In the future, it will be important to move beyond activity alone (as measured by running wheels, photobeam breaks, or computer vision) and try to assay motivation to feed using instrumental behavior assays (nose-poking, lever pressing, etc) (43). If we find that all mice with reduced FAA also have reduced instrumental behavior then we can simply use activity, but we cannot make this correlation until we have more examples of animals lacking FAA.

Author contributions
AS conceived of the manuscript. JT and AS wrote it together. Both authors contributed to the article and approved the submitted version.

Funding
This work was supported by the National Institute of General Medical Sciences of the National Institutes of Health (SC3GM125570 to AS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


In silico functional and structural characterization revealed virulent proteins of Francisella tularensis strain SCHU4

Francisella tularensis is a pathogenic, aerobic gram-negative coccobacillus bacterium. It is the causative agent of tularemia, a rare infectious disease that can attack skin, lungs, eyes, and lymph nodes. The genome of F. tularensis has been sequenced, and ~16% of the proteome is still uncharacterized. Characterizations of these proteins are essential to find new drug targets for better therapeutics. In silico characterization of proteins has become an extremely important approach to determine the functionality of proteins as experimental functional elucidation is unable to keep pace with the current growth of the sequence database. Initially, we have annotated 577 Hypothetical Proteins (HPs) of F. tularensis strain SCHU4 with seven bioinformatics tools which characterized them based on the family, domain and motif. Out of 577 HPs, 119 HPs were annotated by five or more tools and are further screened to predict their virulence properties, subcellular localization, transmembrane helices as well as physicochemical parameters. VirulentPred predicted 66 HPs out of 119 as virulent. These virulent proteins were annotated to find the interacting partner using STRING, and proteins with high confidence interaction scores were used to predict their 3D structures using Phyre2. The three virulent proteins Q5NH99 (phosphoserine phosphatase), Q5NG42 (Cystathionine beta-synthase) and Q5NG83 (Rrf2-type helix turn helix domain) were predicted to involve in modulation of cytoskeletal and innate immunity of host, H2S (hydrogen sulfide) based antibiotic tolerance and nitrite and iron metabolism of bacteria. The above predicted virulent proteins can serve as novel drug targets in the era of antibiotic resistance.

INTRODUCTION
Francisella tularensis, which was first accidentally discovered in 1911, is the causative agent of tularemia and is considered a dangerous potential biological weapon. This bacterium is a small facultative, aerobic, non-spore-forming, non-motile intracellular pathogen that mainly replicates in macrophages [1]. The pneumonic form of tularemia is life-threatening and can result in death. According to the Centers for Disease Control and Prevention (CDC), F.tularensis is a viable biological warfare agent due to its high infectivity, ability to cause death, and low infectious dose [2]. The family Francisellaceae includes three species in the genus Francisella and four subspecies of F. tularensis. Although all have been associated with human disease, only the tularensis and holarctica subspecies of F. tularensis are relatively common [3]. F. tularensis is covered by a capsule that is required for virulence [4,5]. Recently, the presence of Pili (type 4) has been observed which may be the cause of the virulence of the bacteria [6]. F. tularensis has been reported in birds, reptiles, fish, invertebrates, and mammals, including humans. It is caused by contact with infected animals or vectors such as ticks, mosquitoes, and deer flies which makes it a dangerous and highly infectious agent for biological warfare [7][8][9][10] The genome of F. tularensis strain SCHU4 has been sequenced. Its 1.89 Mb base pair long genome with 1910 total genes is contained in only one chromosome. Approximately 83% of the genome is coding which codes about 1667 proteins. Out of these coding proteins, 577 (~16%) are hypothetical proteins (HPs), i.e., there is no experimental evidence regarding the proteins' function and structure, implying that these proteins are yet to be annotated [11,12] Due to the advancements in sequencing technologies, a large amount of protein sequence data is generated that needs to be annotated or characterized but still a significant proportion is demarcated as hypothetical. To keep pace with the exponential growth of sequence data, computational approaches play an important role in deciphering the function of HPs that reduce the laborious experimental protocols [13,14]. These approaches are important for understanding species-specific pathogenicity, new target identification, pathway associations, and evolutionary processes [15][16][17].
Hence, considering the bacterium's pathogenicity and the need to discover a new drug target in the era of antimicrobial resistance, we have computationally annotated uncharacterized proteins of F. tularensis strain SCHU4 using various bioinformatics tools. The study also identified the most virulent proteins from 577 HPs that can serve as novel drug targets.

MATERIALS AND METHODS
Data Retrieval: In this study, hypothetical proteins of F. tularensis strain SCHU4 were retrieved from UniprotKB (https://www.uniprot.org/) [27]. Out of the total 3,733 proteins of F. tularensis, 577 were uncharacterized proteins. The computational workflow used for this study has been illustrated in Figure 1.
Prediction of virulence factor: Virulence factor analysis was done by VirulentPred (http://203.92.44.117/virulent/submit.html), a SVM-based tool that calculates potential virulent proteins and use five-fold cross-validation technique. We have selected default threshold value i.e. 0.0 and score from 0.0 to 1 predict the protein as virulent and minus score indicate nonvirulent protein [45].
Protein-protein interaction prediction and tertiary structure prediction: STRING (Search Tool for the Retrieval of Interacting Genes/Proteins) (https://string-db.org/) database was used to predict the protein-protein interactions of the virulent HPs [46]. Among 66 HPs, we have selected only those proteins which were associated with other known proteins with high confidence score. STRING provides a functional link between proteins called networks along with their scores.
Furthermore, we have determined the 3D structure of those proteins that possess interactions with known proteins with a high confidence score. To predict the protein structure, which is more conserved than protein sequence, we use a protein homology/analogy recognition engine (Phyre 2) (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) that gather homologous sequences against query protein and then scanned against HMM database of known protein structure [47,48]. The structure evaluation was performed by Procheck using SAVES v6.0 server (https://saves.mbi.ucla.edu/) that analyzes the number of residues falling in the allowed region in the Ramachandran Plot as a measure of quality [49].

RESULTS
We have extensively analyzed 577 HPs of F. tularensis using various bioinformatics tools, i.e., CDD, Pfam, SMART, PROSITE, InterPro, CATH, and SVMProt, for domain and/or functional prediction. If the same function is assigned for HPs by five or more tools, then only those HPs were selected for further annotation. Using this strategy, we have identified 119 HPs out of 577. These 119 HPs are further analyzed by using Motif, Superfamily, and Protonet for protein family characterization and were classified as 38 enzymes, 66 binding proteins, 7 receptors, 8 transport proteins as given in Fig. 2 and supplementary Table S2.
Protoparam was used for the prediction of various physiochemical parameters of the filtered 119 proteins. These parameters were molecular weight, aliphatic index, instability index, theoretical pI, etc. >40. The instability index of the protein signifies that the protein is unstable. We identified 28 proteins whose instability index values were greater than 40. The theoretical pI of the HPs lies between 10 and 4. The GRAVY values indicate both hydrophobic (negative) and hydrophilic (positive) HPs [50][51][52]. The Aliphatic Index of the HPs varied between 160 and 59. The physicochemical parameters of 119 HPs are listed in supplementary Table S3. Subcellular location correlates protein biological function as cell offer different chemical environment and interaction [53]. Signal peptides control transport of proteins to target location in all organisms [54,55]. Membrane proteins are involve in signaling between cells, transport and energy transduction [42,43]. Based on the average subcellular prediction results of the three tools HPs were located cytoplasm (40%), membrane (39%), extracellular region (18%), and periplasm (3%). We have predicted presence of signal peptide sequence in 78 HPs. HMMTOP and TMHMM server predicted 65 and 61 proteins as transmembrane helices. These prediction results help to better understanding the new drug target and detail results are mentioned in supplementary Table S4. Prediction of virulent protein is important for identification of novel targets and understanding the complex virulence mechanism of pathogen [45]. VirulentPred predicted 66 HPs as virulent with the score ranges from 0.95 to 1 which is a higher score that minimize false positive hits (supplementary Table S5).
Protein-protein interaction network: We have predicted the association of 66 HPs with other proteins that can further elaborate the role of HPs. Among the complete set of 66 proteins, 25 were dropped out as these did not show any significant interactions. The remaining 41 proteins are related to the known genes with high confidence. We identified interacting partners involved in lipid biosynthesis, signal transduction, transcriptional regulators, iron sulfur binding, extracellular signals, and stimuli. Some proteins interacted with the type 4 pilli, which is suspected to be the cause of the pathogenicity of F. tularensis [6]. Our study found that out of 41 proteins, three proteins Q5NH99, Q5NG42, and Q5NG83, had a very high confidence score of 98%, 95%, and 89.5%, respectively, with their interacting partner. Q5NG83 interacts with the ABC Transporter enzyme, which plays a role in bacterial virulence mainly associated with the uptake of nutrients, while Q5NG42 interacts with GMP synthase required for virulence factor production and Q5NH99 interact with proteins involved in metabolism [56,57]. The STRING results are shown in Figure 3.
Phyre2, a web-based server to predict protein structure based on the principle of homologybased modeling. Query protein is scanned against HMMs proteins of known structure, and topscoring alignments are used to construct crude backbone, and then model is corrected by loop modeling, and side-chain is added to generate the final model [47,48]. We have successfully generated the three best models with high confidence i.e., Q5NG42 protein with 99.9% coverage and 70% confidence, Q5NG83 with 100% confidence and 98% coverage, Q5NH99 with 100% confidence and coverage. The structures predicted by using Phyre2 were evaluated by Procheck to check the quality of the 3D model and more than 90% residues were in favoured region of Ramachandran Plot for all the three predicted structure as shown in Figure 4.

DISCUSSION
Cost-effective sequencing technology has made bacterial genome sequencing affordable to several labs world widely [58]. The published bacterial genome consists of a large group of hypothetical proteins which must be annotated to understand disease pathogenesis, virulent determinants, survival, and propagation. Characterization of HPs by various bioinformatics tools may identify potential biomarkers for the screening and diagnostics of diseases as well as pharmacological targets to design and discover novel drugs [59].
In the present study, we have annotated HPs of F. tularensis by various computational tools to predict the function and structure of proteins that can potentially be involved in the toxicity of the pathogen. The initial step involved identification of conserved region i.e. domain and motif which provide the clue about protein function. We started analysis of 577 HPs using seven functional tools and those HPs are selected for further analysis if the result of each HPs is predicted by five or more tools i.e.>75% prediction analysis which retrieves 119 HPs. These HPs were further annotated for various properties including physicochemical parameters like GRAVY index, aliphatic index, pI, Instability index, cellular localization, transmembrane analysis, functional analysis, and virulent prediction. We then predicted interacting partners of these 66 proteins and based on high confidence score we have selected 3 virulent proteins Q5NH99, Q5NG42 and Q5NG83 with 98%, 95% and 89.5% confidence score respectively (Table 1).
Q5NH99 predicted as phosphoserine phosphatase (PSP), a key essential metabolic enzyme catalyse the dephosphorylation of phosphoserine to serine and inorganic phosphate and is a member of haloacid dehalogenase (HAD) superfamily. PSP have been demonstrated to facilitate entry of Porphyromonas gingivalis into host cells by modulating host cytoskeletal architecture, innate immune responses, dephosphorylating colicin and NF-κ [60][61][62]. Q5NG42, a 196 amino acid length was predicted as Cystathioninebeta-synthase (CBS) catalyzes a condensation reaction to convert homocysteine to cystathionine. This enzyme also produces hydrogen sulfide (H2S) during this catalysis reaction [63]. A number of previous studies in bacterial species have revealed that production of H2S provides antibiotic tolerance and serve as universal defence mechanism . Further supportive evidence was gained by study in which the inactivation of CBS leads to decrease in H2S level and hence more sensitivity towards antibiotics [67]. Hence, CBS can act as a main target against antibiotic resistance in era of antimicrobial resistance (AMR).
Q5NG83 Rrf2-type HTH domain is a 130 residues long DNA binding, helix-turn-helix domain. This domain is present in the proteins that serve as transcription regulators of rrf2 family. Usually, these transcription regulators regulate the genes of bacteria that are involved in nitrite and iron metabolism. For instance, in Escherichia coli one the regulator protein, nsR that contains Rrf2-type HTH domain regulates the NO (nitric oxide) sensitive transcription and thus protects the cell from the NO stress. Similar transcription regulator protein with same role was also found in other bacteria such as Bacillus subtilis and Streptomyces coelicolor whereas Rhizobium leguminosarum contains a transcription regulator protein rirA which is an ironresponsive regulator that involved in uptake of iron [68,69].
Our study has predicted virulent proteins that can play a vital role in future therapeutics of tularemia. These results need to be analyzed in vitro to verify these results experimentally to check the feasibility of these proteins as possible drug targets and their feasibility. These proteins can be essential in determining the virulence mechanism as well as act as the keys for unlocking the mystery related to the immunity of F. tularensis to antibiotics. By further analysis of these proteins, we can also look for binding sites and try to dock leads to these proteins to develop a treatment that has a higher efficacy with fewer side effects.
A recent study by Kumar et al. used pan-genome analysis and genome mining approach to explore the diversity of the genus Francisella. The whole-genome sequence comparisons of the 26 genomes in their study revealed average nucleotide identity (ANI) of 97.0-99.5% for the human pathogens strains cluster, whereas ANI values of 74.2-90.4% were found in the strains that are mainly related to water and environment [70].
Thus the above study shows that there exhibit a good degree of similarity in the coding regions of Francisella genomes. Hence, the functional characterization of HPs in our study may help in the study of proteins of other strains of Francisella